VEGF responsive cell-based assay for determining VEGF bioactivity

ABSTRACT

A VEGF responsive cell-based assay for determining VEGF bioactivity is disclosed. The assay utilizes established signal transduction pathways in a method for determining VEGF bioactivity in a sample.

FIELD OF THE INVENTION

[0001] The present invention generally relates to detection of vascularendothelial growth factor (VEGF) in a sample. Specifically, the presentinvention relates to a VEGF responsive cell-based assay for use in themeasurement of the biological activity of VEGF.

BACKGROUND OF THE INVENTION

[0002] A number of biological properties have been described for VEGF,including the promotion of angiogenesis. Other properties includeendothelial cell migration, endothelial cell proliferation, in vitrocapillary tube formation, inhibition of endothelial cell apoptosis, andincreased in vivo vascular permeability producing edema.

[0003] Because VEGF appears to have a number of significant biologicalproperties, assays for the detection of VEGF and its properties havebecome increasingly important.

[0004] One in vitro bioassay which has been developed is based on theability of human umbilical vein endothelial cells (HUVEC) to migrate inresponse to VEGF. This bioassay includes the steps of first virallytransfecting Rat-2 cells with the consequent production of VEGF protein,and then testing the produced protein extract for the ability tostimulate HUVEC migration. While this assay can be utilized todemonstrate endothelial cell migration stimulated by VEGF protein in theconditioned media of cells, this assay includes a number oftime-consuming steps, which cannot easily be automated.

[0005] Recently, it was proposed that VEGF could be a marker ofcardiovascular disease risk in patients with hypertension. Themeasurement of a patient's VEGF and/or FLT-2 levels may be an indicatorof the effectiveness of a hypertensive therapy.

[0006] Currently, with the seemingly greater importance of VEGF, itwould be both advantageous and desirable to have a VEGF responsive assaythat is less complex than previous assays, that can be automated, andthat is also directly linked to a reporter protein tied to the VEGFreceptor (VEGF-R)/FLK-1 signal transduction pathway.

SUMMARY OF THE INVENTION

[0007] The present invention provides a method for determining VEGFactivity in a sample.

[0008] The present invention also relates to a secondary screen which isuseful for identifying compounds that modulate VEGF receptors.

[0009] The present invention also provides a primary screening mechanismfor screening compounds as inhibitors of VEGF function.

[0010] The present invention also provides a stable cell line for use indetermining VEGF bioactivity and for use in screening compounds whichmodulate VEGF function or VEGF receptor function.

BRIEF DESCRIPTION OF THE FIGURES

[0011] Other advantages of the present invention will be readilyappreciated as the same becomes better understood by reference to thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

[0012]FIG. 1a is a graph illustrating a VEGF responsive assay whereinthe effects of various serum preincubation conditions are shown as afunction of increased luciferase expression wherein cells were seeded 24hours prior to VEGF addition and were harvested 48 hours after VEGFaddition;

[0013]FIG. 1b is a graph illustrating a VEGF responsive assay similar tothat shown in FIG. 1a wherein the concentration of VEGF utilized in theassay was increased to 50 ng/mL as opposed to 25 ng/mL in FIG. 1a;

[0014]FIG. 2 is a graph illustrating the effects of cell density andVEGF₁₂₁ concentration on luciferase expression in a VEGF receptor stabletransfected Hela-Luciferase HLR-ELK-1 cell line;

[0015]FIG. 3 is a graph illustrating VEGF stimulated production ofluciferase in an HLR-ELK-1 cell line wherein the concentration ofVEGF₁₂₁ was varied and the number of cells seeded in each well was keptconstant at approximately 50,000;

[0016]FIG. 4 is a graph illustrating VEGF stimulated production ofluciferase in a VEGF receptor stable transfected HLR-ELK-1 cell line,wherein the number of cells seeded into each well was kept constant atapproximately 50,000, and the concentration of VEGF₁₂₁ was varied;

[0017]FIG. 5 is a graph illustrating the effect of VEGF antibodies onluciferase production in a VEGF receptor cell line;

[0018]FIG. 6 is a graph illustrating luciferase production in aVEGF-receptor (VEGFR) cell line wherein incubation time was compared for24 and 48 hours;

[0019]FIG. 7 is a graph illustrating the optimal VEGF concentration forstimulation of luciferase production in a VEGF-receptor cell line;

[0020]FIG. 8 is a graph illustrating the optimal time for VEGF inducedluciferase production in a VEGF-receptor cell line;

[0021]FIG. 9 is a graph illustrating the effect of compound ZD4190 (aknown VEGF-receptor tyrosine kinase inhibitor) on VEGF stimulation ofluciferase production in a VEGF-receptor cell line;

[0022]FIG. 10 is a graph illustrating luciferase production aftertransfection of a VEGF-receptor cell line with AdVEGF₁₂₁; and

[0023]FIG. 11 is a graph illustrating VEGF stimulated luciferaseproduction using media from AdVEGF₁₂₁ transfected Rat-2 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention provides a VEGF responsive cell-based assayand stable cell line for use therewith for determining VEGF bioactivity.The assay of the present invention allows for the measurement of thebioactivity of VEGF derived from biological samples including plasma,cell culture medium, tissue extracts from tissues or cells transfectedwith VEGF DNA sequences, or combinations thereof. The method of thepresent invention can also be adapted for use in high throughputscreening and in secondary screens to identify novel small moleculemodulators (inhibitors or activators) of a VEGF receptor, specificallyFLK-1.

[0025] The assay of the present invention utilizes a stable VEGFresponsive cell line which comprises HeLa cells which have been stablytransfected with a reporter vector having an expressible reporterelement and a DNA binding element disposed adjacent thereto. Preferably,the reporter vector includes a gene encoding a detectable gene productwhich is disposed downstream of a basic promoter element, preferably aTATA box, which is joined to the binding element which is preferably aGAL4 binding element. The stable cell line is also transfected with avector encoding a CMV promoter-driven ELK-1 transcription factor (whichis tied to the MAP kinase pathway) and is fused to a yeast GAL4 DNAbinding domain and a yeast GAL4 binding element-driven luciferasereporter construct. A third vector encoding a gene capable of expressingmouse FLK-1 VEGF receptor is also transfected into the cells and thestable cell line generated therefrom can be utilized to demonstrateupregulation for the detectable gene product (luciferase expression) inthe presence of VEGF. That is, utilizing established signal transductionpathways, VEGF bioactivity can be assayed.

[0026] In general, utilizing known signal transduction relationshipsand/or pathways, a sample to be assayed for VEGF bioactivity is placedin a container containing the stable cell line as described above. IfVEGF is present in the sample, VEGF activates FLK-1 expressed by thestable cell line. Activated FLK-1, which is a known VEGF receptor, thenactivates MAP kinase (Kroll and Waltenberger, J. Biol. Chem.,1997:272:32521-32527; Doanes et. al., Biochem. Biophys. Res. Comm.,1999;255:545-548. The activated MAP kinase phosphorylates the fusiontrans-activation protein (GAL4 DNA binding domain [dbd] fused withELK-1). The phosphorylated fusion protein binds to the GAL4 DNA bindingsite of the reported vector activating luciferase expression. Luciferaseexpression can be detected utilizing techniques well-known in the art.The presence or expression of luciferase indicates VEGF activity in thesample.

EXAMPLES

[0027] The following examples further illustrate the present invention.The examples are intended merely to be illustrative of the presentinvention and are not to be construed as being limited.

METHODS

[0028] Cell Line Production

[0029] HeLa cells stably transfected with the GAL4 luciferase reporterand a vector expressing a fusion protein composed of the GAL4 DNAbinding domain and the transactivation domain of the transcriptionfactor ELK-1 (GAL4-ELK-1-fusion) were purchased from Stratagene Inc.These cells were co-transfected with a CMV driven FLK-1 expressionvector (licensed from The Ludwig Institute, Germany) and a Zeocinselection expression vector (pcDNA3.1/zeo (+), #V860-20) obtained fromInvitrogen in the HeLa cells. After appropriate antibiotic selection,stable transfectants were identified that respond to VEGF by increasingluciferase expression.

[0030] Cell Culture and Luciferase Assay

[0031] The optimal transfected VEGF-receptor cell line (referred to asclone #5) was maintained in Dulbecco's Modified Eagle Medium (LifeTechnologies, Gaithersburg, Md.) containing 10% fetal bovine serum(FBS), Geneticin (250 μg/mL), Hygromysoin B (100 μg/mL), and Zeocin (100μg/mL). Cells were seeded into 24-well culture plates and allowed toattach prior to the stimulation of luciferase production by the additionof VEGF₁₂₁ (298-VS-005, R&D Systems, Minneapolis, Md.) at the indicatedconcentrations. Luciferase activity was measured 24 to 48 hourspoststimulation as outlined in the technical insert for the luciferaseassay system available from Promega Corporation (E1501, Madison, Wis.).The cells were washed with PBS before being lysed in 200 μL of 1Xreporter lysis buffer. The entire plate was frozen at −80° C. toincrease cell lysis. Upon thawing, 50 μL of the cell extract wastransferred into a 96-well plate. Luciferase assay reagent (100 μL) wasauto-injected, and light production was measured using a microplateluminometer.

EXAMPLE 1

[0032] Effect of Serum Preincubation Conditions on Luciferase Activity

[0033] Cells were seeded into wells 24 hours prior to VEGF addition. Thecells were harvested 48 hours after the addition of 25 ng/mL of VEGF.Cells were pre-incubated under various serum conditions: (1) 10% FBS,(2) 0.2% FBS, (3) serum free, and (4) nutridoma, a serum-free mediasupplement. The results are shown in FIG. 1a. The optimal serumpre-incubation condition was found to be preincubation in 10% FBS. Anidentified experiment was performed except that the concentration ofVEGF added was increased to 50 ng/mL. The results are shown in FIG. 1b.The increased concentrations of VEGF was found to increase luciferaseexpression.

EXAMPLE 2

[0034] Effects of Cell Density and VEGF₁₂₁ Concentration on LuciferaseExpression

[0035] Cells were prepared as described above. Cells were seeded intowells 24 hours prior to the addition of VEGF₁₂₁. VEGF₁₂₁ was added tothe cells at concentrations of 25 and 50 ng/mL, respectively. Cellsseeded into the wells were tested at densities of: 50, 100, 150, and 200K/well. The results are shown in FIG. 2.

EXAMPLE 3

[0036] Dose Response to VEGF in the Parental Cell Line (HLR-ELK-1)

[0037] Cells were prepared as described above. 50 K/well were seeded andkept in 10% FBS throughout the experiment. VEGF₁₂₁ was added 24 hoursafter seeding, and the cells were harvested and analyzed 48 hours afterthe addition of VEGF₁₂₁. The results are shown in FIG. 3. The resultsshowed that the parental cell line was no more responsive to VEGF₁₂₁stimulation than the control.

EXAMPLE 4

[0038] Dose Response of VEGF-Receptor Stable Transfected HLR-ELK1 CellLine

[0039] Cells were prepared as described above. Cells were transfectedwith the plasmid vector expressing the FLK-1 VEGF receptor were seededat 50 K/well and maintained in 10% FBS throughout the experiment.VEGF₁₂₁ was added 24 hours after seeding, and the cells were harvested48 hours after the addition of VEGF₁₂₁. The concentration of VEGF₁₂₁ wastested at 10, 25, 50, 75, 100, and 150 ng/mL. Maximum increase inluciferase expression was found at a VEGF₁₂₁ concentration of 100 ng/mLas shown in FIG. 4.

EXAMPLE 5

[0040] Dose Responsive Specificity of the VEGF-Receptor Cell Line

[0041] To demonstrate the dose responsive specificity of theVEGF-receptor cell line, cells were prepared as described above. Thetransfected cells were incubated with VEGF₁₂₁, the adenovirus NULLvector (AdCLX), antihuman VEGF₁₂₁ antibodies (AF-293-NA [polyclonal],MAB293[monoclonal], R&D Systems), a VEGF receptor-specific tyrosinekinase inhibitor (ZD4190, Zeneca [Wedge S. R., Ogilvie D. J. Inhibitionof VEGF signal transduction: Identification of ZD4190. Adv. Exp. Med.Biol., 2000;476(Angiogenesis: From the Molecular to IntegrativePharmacology):307-310; and Wedge S. R., Ogilvie D., Dukes M., KendrewJ., Curwen J. O., Hennequin L. F., Thomas A. P., et. al., ZD4190: Anorally active inhibitor of vascular endothelial growth factor signalingwith broad-spectrum antitumor efficacy. Cancer Res.,2000;60(4):970-975]), media from ADVEGF₁₂₁ transfected rat 2 cells atvarious concentrations, or the VEGF-receptor cell line was directlyinfected with an adenovirus containing VEGF₁₂₁ (AdGV121.10, CI-1023,GenVec, Inc., Rockville, Md.) at various concentrations.

[0042] Cells were seeded at 50 K/well 24 hours prior to the addition ofVEGF/anti-VEGF and were collected 48 hours later. Referring to FIG. 5,the results for the antihuman VEGF antibodies are shown. The polyclonalantibodies did not significantly affect luciferase expression. Themonoclonal antibodies at the 1:100 and 1:1000 dilutions affected theluciferase expression.

[0043]FIG. 9 shows the effects of the known VEGF receptor tyrosinekinase inhibitor (ZD4190) on luciferase expression. The VEGF receptortyrosine kinase inhibitor affected luciferase expression in adose-response manner.

[0044]FIG. 10 shows the effects of AdVEGF121 obtained from using a mediafrom AdVEGF₁₂₁ transfected rat 2 cells. The addition of AdVEGF₁₂₁ to theVEGF-receptor cell line affected luciferase expression in a closeresponse manner both from the AdVEGF₁₂₁ itself and from the media fromAdVEGF₁₂₁ transfected rat 2 cells.

EXAMPLE 6

[0045] Luciserase Production in VEGF-Receptor Cell Line at 24 and 48Hours After the Addition of VEGF

[0046] Cells were prepared as described above. VEGF₁₂₁ was added tocells (50 K/well) 24 hours after seeding. VEGF₁₂₁ was added to the cellsat concentrations of either 25 or 50 ng/mL. Luciferase expression wasmeasured 24 hours after the addition of VEGF₁₂₁ and 48 hours after theaddition of VEGF₁₂₁. The results are shown in FIG. 6. Maximum luciferaseexpression was found in the cells treated with 50 ng/mL of VEGF₁₂₁ at 24hours post-VEGF₁₂₁ introduction.

EXAMPLE 7

[0047] Determination of Optimal VEGF Concentration

[0048] VEGF-receptor cells were seeded at 50 K/well and incubated in 10%FBS throughout the experiment. VEGF₁₂₁ was added to the cells 24 hoursafter seeding, and the cells were harvested 18 hours later. The VEGF₁₂₁was applied to the cells at the following concentrations: 1, 5, 10, and200 ng/mL. The results are shown in FIG. 7. The optimal VEGF₁₂₁concentration ranges from approximately 50 ng/mL to approximately 200ng/mL.

EXAMPLE 8

[0049] Optimal Incubation Time for VEGF-Induced Luciferase Production

[0050] Cells were prepared as described above VEGF-receptor cells wereseeded at 50 K/well and incubated for 24 hours in 10% FBS prior to theaddition of VEGF₁₂₁. Cells were harvested at 0.5, 1, 2, 3, 4, 5, 6, 7,8, 18, and 24 hours, after VEGF₁₂₁ (25 ng/mL) addition. FIG. 8 shows theresults of this experiment. Approximately 18 hours of expression timewas found to yield maximum luciferase expression (production).

[0051] All publications mentioned in the specification are hereinincorporated by reference to the same extent as if each independentpublication was specifically and individually indicated to beincorporated by reference.

[0052] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications. The application is intended to cover anyvariations, uses, or adaptations following, in general, the principlesof the invention and including such departures from the presentdisclosure within known or customary practice within the art to whichthe invention pertains and may be applied to the essential featuresherein before set forth.

What is claimed is:
 1. A method for determining vascular endothelialgrowth factor (VEGF) activity in a sample, said method comprising thesteps of: Contacting a sample to be assayed for VEGF activity with astable cell line comprising cells transfected with a reporter vectorhaving an expressible reporter element and a DNA binding site disposedadjacent thereto, a chimeric transactivatable vector comprising a geneencoding a phosphorylatable protein and a DNA binding domain whichspecifically binds to the DNA binding site, and an expression vectorencoding a gene for a VEGF receptor detecting the presence of expressedreporter element indicating VEGF activity; and Detecting expression ofthe reporter element, wherein expression of the reporter elementindicates VEGF activity.
 2. A method according to claim 1, wherein thereporter vector further comprises a GAL4 binding element.
 3. A methodaccording to claim 2, wherein the reporter vector comprises a geneencoding for a detectable product.
 4. A method according to claim 3,wherein the detectable product comprises luciferase.
 5. A methodaccording to claim 3, wherein the gene encoding for the detectableproduct is operably linked to a promoter element.
 6. A method accordingto claim 5, wherein the promoter element comprises a TATA box.
 7. Amethod according to claim 1, wherein the phosphorylatable proteinencoded by chimeric transactivatable vector can be phophorylated byMAPK.
 8. A method according to claim 1, wherein the phosphorylatableprotein comprises ELK-1.
 9. A method according to claim 1, wherein thegene encoding for the phosphorylatable protein is operably linked to apromoting element.
 10. A method according to claim 1, wherein VEGFreceptor comprises FLK-1.
 11. A method according to claim 1, wherein theVEGF receptor encoding gene is operably linked to a promoter element.12. A method according to claim 11, wherein the VEGF receptor encodinggene is FLK-1.
 13. A method according to claim 1, wherein the stablecell line comprises HeLa cells.
 14. A method according to claim 1,wherein said contacting step further comprises binding VEGF present inthe sample with expressed VEGF receptor.
 15. A method according to claim14, wherein said including contacting step further comprises activatingMAPK with the expressed VEGF receptor.
 16. A method according to claim15, further comprising the step of expressing the trans-activator vectorto produce a chimeric product comprising the phosphorylatable proteinand DNA binding domain.
 17. A method according to claim 16, furthercomprising the step of phosphorylating the chimeric product with theactivated MAPK.
 18. A method according to claim 17, further comprisingthe step of binding the phosphorylated chimeric product to the DNAbinding site of the reporter vector, wherein expression of theexpressible reporter element is activated indicating the presence ofVEGF in the sample.
 19. A method according to claim 1, wherein thesample comprises biological fluids.
 20. A method according to claim 19,wherein the biological fluids comprise plasma or cell culture media. 21.A method according to claim 1, wherein the sample comprises cells,tissue, tissue extracts, and combinations thereof.
 22. A methodaccording to claim 1, wherein the VEGF activity is detectable in aconcentration >1 mg/mL.
 23. A method according to claim 1, wherein theVEGF activity is detectable in a concentration range from approximately1 ng/mL to approximately 200 ng/mL.
 24. A method according to claim 1,further comprising the step of incubating the sample with stable cellline for a period of time ranging from approximately 4 hours toapproximately 24 hours.
 25. A method according to claim 1, furthercomprising the step of incubating the sample with stable cell line for aperiod of time ranging from approximately 10 hours to approximately 20hours.
 26. A method of determining whether a candidate compound isuseful for modulating VEGF receptor activity, said method comprising thesteps of: (a) providing a cell expressing the VEGF receptor FLK-1; (b)contacting the cell with a candidate compound; (c) measuring VEGFreceptor activity, wherein the altered VEGF receptor activity relativeto a cell not contacted with the candidate compound, indicates that thecandidate compound modulates VEGF receptor activity.
 27. A methodaccording to claim 26, wherein the cell further comprises a reportervector having an expressible reporter element and a DNA binding sitedisposed adjacent thereto, and a chimeric transactivator vectorcomprising a gene encoding a phosphorylatable protein and a DNA bindingdomain which specifically binds to the DNA binding site.
 28. A methodaccording to claim 27, wherein said measuring step is further defined ascomparing levels of the expressed reporter element from the cellcontacted with the candidate compound relative to a cell not contactedwith the candidate compound.
 29. A method for determining whether acandidate compound is useful for modulating VEGF activity, said methodcomprising the steps of: (a) providing a cell expressing VEGF; (b)contacting the cell with a candidate compound; (c) contacting a sampleto be assayed for VEGF activity with a stable cell line comprising cellstransfected with a reporter vector having an expressible reporterelement and a DNA binding site disposed adjacent thereto, a chimerictransactivatable vector comprising a gene encoding a phosphorylatableprotein and a DNA binding domain which specifically binds to the DNAbinding site, and an expression vector encoding a gene for a VEGFreceptor detecting the presence of expressed reporter element indicatingVEGF activity; and (d) Detecting expression of the reporter element,wherein expression of the reporter element indicates VEGF activity.wherein altered VEGF activity relative to a cell not contacted with thecandidate compound indicates that the candidate compound modulates VEGFactivity.
 30. A method according to claim 27, wherein the reportervector further comprises a GAL4 binding element.
 31. A method accordingto claim 30, wherein the reporter vector comprises a gene encoding for adetectable product.
 32. A method according to claim 31, wherein thedetectable product comprises luciferase.
 33. A method according to claim31, wherein the gene encoding for the detectable product is operablylinked to a promoter element.
 34. A method according to claim 33,wherein the promoter element comprises a TATA box.
 35. A methodaccording to claim 29, wherein the phosphorylatable protein encoded bychimeric trans-activatable vector can be phophorylated by MAPK.
 36. Amethod according to claim 29, wherein the phosphorylatable proteincomprises ELK-1.
 37. A method according to claim 29, wherein the geneencoding for the phosphorylatable protein is operably linked to apromoting element.
 38. A method according to claim 29, wherein VEGFreceptor comprises FLK-1.
 39. A method according to claim 29, whereinthe VEGF receptor encoding gene is operably linked to a promoterelement.
 40. A method according to claim 39, wherein the VEGF receptorencoding gene is FLK-1.
 41. A method according to claim 29, wherein thestable cell line comprises HeLa cells.
 42. A method according to claim29, wherein said contacting step further comprises binding VEGF presentin the sample with expressed VEGF receptor.
 43. A method according toclaim 42, wherein said including contacting step further comprisesactivating MAPK with the expressed VEGF receptor.
 44. A method accordingto claim 43, further comprising the step of expressing thetransactivator vector to produce a chimeric product comprising thephosphorylatable protein and DNA binding domain.
 45. A method accordingto claim 44, further comprising the step of phosphorylating the chimericproduct with the activated MAPK.
 46. A method according to claim 45,further comprising the step of binding the phosphorylated chimericproduct to the DNA binding site of the reporter vector, whereinexpression of the expressible reporter element is activated indicatingthe presence of VEGF in the sample.
 47. A method according to claim 29,wherein the sample comprises biological fluids.
 48. A method accordingto claim 47, wherein the biological fluids comprise plasma or cellculture media.
 49. A method according to claim 29, wherein the samplecomprises cells, tissue, tissue extracts, and combinations thereof. 50.A stable cell line transfected with a reporter vector encoding aluciferase gene and a GAL4 DNA binding site; a chimeric transactivatorvector encoding for an ELK-1/GAL4 DNA binding domain fusion protein; anda vector encoding for VEGF receptor FLK-1.